Genetic studies have yielded conflicting results as to whether DRD2-Taq IA polymorphism is associated with addiction as well as other neurobiological traits and neuropsychiatric disorders. The variant resides in the DRD2 neighboring gene, ANKK1. Recently, evidence has been replicated that ANKK1 has more significant association with nicotine and alcohol dependences than DRD2 does. However, the function of ANKK1 is largely unknown. Purposes: I will implement transcripteomic, proteomic, and bioinformatic analyses to characterize function of ANKK1 and its genetic variants, and to evaluate the interaction between ANKK1 variants and an environmental factor - nicotine stimulation. Methods: On the basis of previous genetic association studies and protein structural analyses, I will select two variants in ANKK1 as subjects: the well- known Taq IA (rs1800497) and a likely centromeric functional rs2734849, in addition to a kinase-less mutant as a negative control. I will magnify ANKK1 function in the genomic response by over-expression and utilize high-throughput microarray assay to profile the differential genome-wide gene expression, 1) caused by ANKK1 kinase activity in a comparison of ANKK1 with the kinase-less mutant;2) altered by ANKK1 genetic variation in a comparison of two alleles of a polymorphism;and 3) induced by nicotine stimulation in a comparison of ANKK1-transfected cells treated with or without nicotine. Furthermore, I will employ protein pull- down assay to identify differential ANKK1-interacting proteins and employ reverse in-gel kinase assay to identify differential kinase substrates of ANKK1 variants, in a combination with MALDI-TOF mass spectrometry. Finally, I will implement three complementary bioinformatic tools, GOEAST, GSEA and IPA, to integrate all the direct and indirect evidence collected into the ANKK1-relevant cell signaling pathways and protein/gene interaction networks. I will evaluate the contribution of ANKK1 genetic variants and nicotine stimulation to the genomic response, as well as the interplay of ANKK1 genetic variants with nicotine stimulation. In this proposal, I will utilize simple, easy-to-perform, and high-throughput techniques as well as comprehensive and complementary data analysis and data mining to ensure the feasibility and effectiveness of this project. Outcomes: Accomplishment of this proposal will provide functional information about ANKK1 kinase activity, ANKK1 genetic variants, and ANKK1 role in nicotine stimulation. The established ANKK1- relevant signaling pathways, influenced by ANKK1 genetic variants and nicotine stimulation, will provide a global and dynamic picture of ANKK1 function in cell signaling and regulatory events. I expect the results will also provide an explanation to the controversial association studies of the DRD2-Taq IA polymorphism. Benefits: If successful, the approach could provide a good example of studying genotype-by-environment interaction in a cell model. In the long term, elaboration of the ANKK1-relevant signaling pathways might pinpoint a potential target(s) for developing therapeutic strategies to prevent or to treat addiction. PUBLIC HEALTH RELEVANCE: Project Narrative This study is to elucidate the function of ANKK1, a serine/threonine kinase, in cell signaling and regulatory events and to evaluate the genotype-by-environment interaction between ANKK1 genetic variants and nicotine stimulation. If successful, this basic research should provide significant information about genetic and environmental perturbations of intracellular signaling pathways and gene regulation networks, which would shed light on understanding of the molecular mechanisms underlying addiction.